Compositions and methods comprising serratia peptidase for inhibition and treatment of biofilms related to certain conditions

ABSTRACT

Physiologically acceptable anti-biofilm compositions comprising  Serratia  peptidase and optionally one or more of bromelain, papain and a fibrinolytic enzyme. Additional components can include antimicrobials, antibiotics, antifungals, herbals, chelating agents, lactoferrin and related compounds, minerals, surfactants, binders, and fillers useful for the inhibition and treatment of gastrointestinal biofilms in humans. Physiologically acceptable anti-biofilm compositions containing these enzymes are useful in the inhibition, reduction and/or treatment of biofilms such as in the ear, vagina, joints, bones, gut, surgical sites and other locations, and are useful for the inhibition, reduction and/or treatment of associated systemic symptoms caused by biofilm associated microorganisms.

PRIORITY CLAIM

The present application claims the benefit of copending U.S. ProvisionalPatent Application Ser. No. 61/263,776, filed Nov. 23, 2009, whichapplication is incorporated herein by reference in its entirety.

BACKGROUND

A “biofilm” is a well known phenomenon and may be defined as apopulation of microbial cells growing on a surface and enclosed in aself-produced matrix of extracellular polymeric material, which mediatesadhesion of the cells to each other and to surfaces. Biofilms are notsimply passive assemblages of cells that are stuck to surfaces, but arestructurally and dynamically complex biological systems. As comparedwith cells that are planktonic in nature, bacteria growing in biofilmsexhibit a different phenotype with respect to growth rate and genetranscription. See http://en.wikipedia.org/wiki/Biofilm.

Unwanted biofilms have been responsible, for example, for the fouling ofcooling-water towers, water pipelines, membrane units andfood-processing plants. Biofilms are notoriously difficult to eradicate.Microbes in industrial biofilms are protected from antimicrobialchemicals, environmental bacteriophages, and phagocytic amoebae. (DonlanR M, Costerton J W. Biofilms: survival mechanisms of clinically relevantmicroorganisms. Clin Microbiol Rev 2002;15167-293.)

In addition to their importance in industry, biofilms may be involved ina significant percentage of human microbial infections (Potera C.Forging a link between biofilms and disease. Science 1999;283:1837-8).Parsek and Singh proposed four criteria for defining a biofilm etiologyof an infection: the pathogenic bacteria are surface associated oradherent to a substratum; direct examination reveals bacteria inclusters, encased in a matrix of bacterial or host constituents; theinfection is localized; and the infection is resistant to antibiotictherapy despite the antibiotic sensitivity of the constituent planktonicorganisms (Parsek M R, Singh P K. Bacterial biofilms: an emerging linkto disease pathogenesis. Annu Rev Microbiol 2003;57:677-701.)

Biofilm infections can be involved in the etiology of dental caries,periodontal disease, cystic fibrosis (CF) airway infections, nativevalve endocarditis, chronic prostatitis, otitis media, and vaginalinfections. Biofilm microorganisms are also involved in implant-relatedinfections, in which adherent microbial populations form on the surfacesof catheters, prosthetic heart valves, joint replacements, and otherdevices (Donlan R M. Biofilms and device-associated infections. EmergInfect Dis 2001;7:277-81.)

The intestinal tract provides a reservoir for many antibiotic-resistantbiofilm bacteria, including Enterobacteriaceae species, Pseudomonasaeruginosa, and Acinetobacter species (Donskey C J. The role of theintestinal tract as a reservoir and source for transmission ofnosocomial pathogens. Clin Infect Dis 2004;39:219-26.) The humanopportunistic pathogen, Pseudomonas aeruginosa, is a major cause ofinfection-related mortality among the critically ill patients, andcarries one of the highest case fatality rates of all gram-negativeinfections. Although the lungs have been traditionally considered to bea major site of P. aeruginosa infection among critically ill patients, asignificant number of these infections arise as a result of directcontamination of the airways by the gastrointestinal flora or byhematogenous dissemination from the intestines to the lung parenchyma.Effective methods for the inhibition, reduction and/or treatment of P.aeruginosa would have a significant impact for this condition.

With respect to biofilms in the gut, it is now known that bacteria canexist for example as biofilms on the intestinal epithelium, within themucus layer covering it, and on food particles in the lumen. (MacFarlaneS, MacFarlane G T. Composition and metabolic activities of bacterialbiofilms colonizing food residues in the gastrointestinal tract. ApplEnviron Microbiol 2006;72:6204-11; Probert H M, Gibson G R. Bacterialbiofilms in the human gastrointestinal tract. Curr Issues IntestMicrobiol 2002;3:23-7.) Gastrointestinal biofilm-associated bacteriainclude Bacteroides ssp., Clostridium ssp., Fusobacterium ssp.,Klebsiella ssp., Spirochaetes ssp., Pseudomonas aeriginosa, Escherichiacoli, Helicobacter pylori, Bifidobacterium ssp., and gram-positivecocci.

Thus, there has gone unmet a need for improved methods, compositions,etc., related to reduction of biofilms within the ear, vagina, joints,bones, gut, surgical sites and other locations in mammals. The presentmethods, etc., provide one or more of these and/or other advantages.

SUMMARY

The present compositions, medicaments, therapeutics, systems, methods,etc., are directed to the reduction or inhibition of harmful biofilm(s)in animals, for example biofilms occurring in conjunction with certaindiseases or conditions including bacterial vaginosis, bacterialvaginitis or fungal vaginitis (i.e., inflammations of the vagina due tobacteria or fungi); osteomyelitis; otitis media; chronic sinusitis;chronic prostatitis, native valve endocarditis; biofilm on a mucosalsurface; and, biofilm infections of medical implants and medicaldevices. The compositions include physiologically acceptableanti-biofilm compositions comprising at least Serratia peptidase in atherapeutic amount. In some embodiments, the compositions furthercomprise therapeutic amounts of one or more of bromelain, papain, and afibrinolytic enzyme. The fibrinolytic enzyme can be, for example,nattokinase, lumbrokinase or Fusarium protease. Fusarium protease is afibrinolytic enzyme that has been reported to be more potent thannattokinase in its fibrinolytic activity.

The compositions are administered to the patient for a time sufficientto cause significant biofilm reduction on the target site, such as amucosal surface, in the mammal. The compositions are administered, forexample, as nutraceutical, therapeutic, or pharmaceutical compositions,and are typically suitable for oral ingestion by or topical applicationto mammals such as humans. The current discussion also includes methodsof making and using or administering such compositions.

In another aspect, the present physiologically acceptable anti-biofilmcompositions, methods, etc., are also directed to the use of digestiveenzymes for the inhibition and reduction of pathogenic biofilm in thegastrointestinal tract of humans.

For example, the physiologically acceptable anti-biofilm compositions,methods, etc., can be directed to the use of cellulases, hemicellulases,lysozyme, pectinases, amylases, DNase I, β-1,6-N-acetylglucosaminidase,and other hydrolases that are capable of digesting theexopolysaccharide, exoprotein, and nucleotide matrix of biofilms.

The present physiologically acceptable anti-biofilm compositions,methods, etc., are also directed to oral physiologically acceptableanti-biofilm compositions for the inhibition and treatment of pathogenicgastrointestinal biofilms in humans.

In certain embodiments, the present physiologically acceptableanti-biofilm compositions, methods, etc., are directed to agents thatare foodborne, waterborne or are nosocomial. Some embodiments arefurther directed to biofilm infections that are antibiotic-resistantand/or recurrent. The physiologically acceptable anti-biofilmcompositions, etc., may be used in conjunction with antibiotics orantimicrobials. In addition these physiologically acceptableanti-biofilm compositions may be used in patients whose biofilminfections have failed to respond to antibiotics or antimicrobials.

The present physiologically acceptable anti-biofilm compositions,methods, etc., are also directed to the inhibition and treatment ofbiofilm infections caused by bioterrorist agents.

Thus, in one aspect, the present compositions, methods, etc., aredirected to a physiologically acceptable anti-biofilm compositionsuitable for administration to a mammal, the composition comprising atleast one pharmaceutically acceptable carrier and Serratia peptidase inamounts capable of significant biofilm degradation in the mammal uponadministration to the mammal.

In some embodiments, the present compositions, methods, etc., furthercomprise one or more of bromelain, papain and a fibrinolytic enzyme. Thefibrinolytic enzyme can comprise at least one of nattokinase orlumbrokinase, and the composition can be configured for oraladministration such that the composition can capable of gastrointestinalabsorption while retaining the anti-biofilm activity after passingthrough the stomach. The composition can also be administered via anyother suitable route, such as topically, and other indirect or directroutes, such as buccal/sublingual, rectal, oral, nasal, vaginal,pulmonary, intraperitoneal, subcutaneous, intranasal, or intravenous.

The compositions, methods, etc., further can comprise at least onechelating agent capable of chelating at least one of calcium or ironconfigured for administration in an amount capable of significantbiofilm degradation in the mammal. The chelating agent can be at leastone of lactoferrin or a lactoferrin peptide capable of chelation.

The compositions, methods, etc., further can comprise at least one of ananti-biofilm acid-stable cellulase or an anti-biofilm anti-polymericβ-,6-N-acetyl-D-glucosamine (poly-β-,6-GlcNAc) agent, or at least one ofan acid-stable hemicellulase/pectinase complex, β-gluconase, acidprotease, or alkaline protease. The composition further can comprise atleast one acid-stable agent selected from the following: adisaccharidase; amylase; α-amylase; β-amylase; glucoamylase;endoglucanase; xylanase; lipase; lysozyme; an enzyme with dipeptidylpeptidase IV (DPP-IV) activity; chitosanase; ficin; kiwi protease; anyplant-derived protease or proteinase, or phytase. The compositionfurther can comprise at least one acid-stable enzyme configured foradministration in an amount capable of significant biofilm degradationin the mammal, the at least one enzyme selected from the following:1,2-1,3-α-D-mannan mannohydrolase, 1,3-β-D-xylanxylanohydrolase,1,3-β-D-glucan glucanohydrolase, 1,3(1,3;1,4)-α-D-glucan3-glucanohydrolase, 1,3(1,3;1,4)-β-D-glucan 3(4)-glucanohydrolase,1,3-1,4-α-D-glucan 4-glucanohydrolase, 1,4-α-D-glucan glucanehydrolase,1,4-α-D-glucan glucohydrolase, 1,4-(1,3:1,4)-β-D-glucan4-glucanohydrolase, 1,4-β-D-glucan glucohydrolase, 1,4-β-D-xylanxylanohydrolase, 1,4-β-D-mannan mannanohydrolase, 1,5-α-L-arabinanohydrolase, 1,4-α-D-glucan maltohydrolase, 1,6-α-D-glucan6-glucanohydrolase, 2,6-β-fructan fructanohydrolase, α-dextrin6-glucanohydrolase, α-D-galactoside galactohydrolase, α-D-glucosideglucohydrolase, α-D-mannoside mannohydrolase, acylneuraminyl hydrolase,Aerobactercapsular-polysaccharide galactohydrolase, β-D-fructofuranosidefructohydrolase, β-D-fucoside fucohydrolase, α-D-fructanfructohydrolase, β-D-galactoside galactohydrolase, β-D-glucosideglucohydrolase, β-D-glucuronoside, glucuronosohydrolase, β-D-mannosidemannohydrolase, β-N-acetyl-D-hexosaminide N-acetylhexosamino hydrolase,cellulose-sulfate sulfohydrolase, collagenase, dextrin6-α-D-glucanohydrolase, glycoprotein-phosphatidylinositolphosphatidohydrolase, hyaluronate 4-glycanohydrolase,hyaluronoglucuronidase, pectin pectylhydrolase, peptidoglycanN-acetylmuramoylhydrolase, phosphatidylcholine 2-acylhydrolase,phosphatidylcholine 1-acylhydrolase, poly(1,4-α-D-galacturonide),poly(1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolase, proteases,sucrose α-glucosidase, triacylglycerol acylhydrolase, triacylglycerolprotein-acylhydrolase.

The compositions, methods, etc., also can comprise a green tea extract,an acid-stable subtilisin or an acid-stable DNAse I, a chelating agentselected from the group comprising ethylenediamine-N,N,N′,N′-tetraaceticacid (EDTA); the disodium, trisodium, tetrasodium, dipotassium,tripotassium, dilithium and diammonium salts of EDTA; the barium,calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum,magnesium, manganese, nickel, samarium, strontium, and zinc chelates ofEDTA; trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraaceticacidmonohydrate; N,N-bis(2-hydroxyethyl)glycine;1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid;1,3-diaminopropane-N,N,N′,N′-tetraacetic acid;ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionicacid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonicacid)hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triaceticacid; ethylenediamine-N,N,N′,N′-tetrakis(methylenephosponic acid);O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid;N,N-bis(2-hydroxybenzyl)ethylene diamine-N,N-diacetic acid;1,6-hexamethylenediamine-N,N,N′,N′-tetraacetic acid;N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid;1,2-diaminopropane-N,N,N′,N′-tetraacetic acid; nitrilotriacetic acid;nitrilotripropionic acid; the trisodium salt ofnitrilotris(methylenephosphoric acid);7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontanehexahydrobromide; triethylenetetraminie-N,N,N′,N″,N″′,N″′-hexaaceticacid; deferoxamine; deferiprone; and deferasirox.

The compositions, methods, etc., further can comprise, or exclude, anantibiotic, and can comprise quercetin, seaprose or Fusarium protease.

Another aspect herein is directed to methods of inhibiting a biofilminfection in a mammal comprising: identifying the presence of thebiofilm infection, administering to the mammal a therapeuticallyeffective amount of a composition comprising at least onepharmaceutically acceptable carrier and Serratia peptidase, bromelain,papain and a fibrinolytic enzyme in amounts capable of significantbiofilm degradation in the mammal, in an amount and for a timesufficient to cause significant biofilm degradation within the mammal.

The methods further can comprise identifying the presence of the biofilminfection in the gastrointestinal system of the mammal, and orallyadministering to the mammal the therapeutically effective amount of thecomposition in an amount and for a time sufficient to cause significantbiofilm degradation within the gut of the mammal.

The methods further can comprise identifying the presence of the biofilminfection at a surface of a body part of the mammal other than thegastrointestinal system, and topically administering to the surface ofthe body part the therapeutically effective amount of the composition inan amount and for a time sufficient to cause significant biofilmdegradation at the surface of the body part. The surface can be forexample exposed skin or an internal surface of the mammal. As withcertain other methods and compositions herein, the methods can alsocomprise other routes and/or targets of administration.

The methods further can comprise administering at least one oflactoferrin and a chelating agent; at least one of an anti-biofilmacid-stable cellulase or an anti-biofilm anti-polymericβ-1,6-N-acetyl-D-glucosamine (poly-β-1,6-GlcNAc) agent; or, at least oneof an acid-stable hemicellulase/pectinase complex, β-gluconase, acidprotease, or alkaline protease in an amount and for a time sufficient tocause significant biofilm degradation within of the mammal.

The methods further can comprise administering at least one anacid-stable agent, for example selected from the following: adisaccharidase; amylase; α-amylase; β-amylase; glucoamylase;endoglucanase; xylanase; lipase; lysozyme; an enzyme with dipeptidylpeptidase IV (DPP-IV) activity; chitosanase; ficin; kiwi protease; anyplant-derived protease or proteinase, or phytase. The methods also cancomprise administering at least one an acid-stable selected from thefollowing: 1,2-1,3-α-D-mannan mannohydrolase,1,3-β-D-xylanxylanohydrolase, 1,3 -β-D-glucan glucanohydrolase,1,3(1,3;1,4)-α-D-glucan 3-glucanohydrolase, 1,3(1,3;1,4)-β-D-glucan3(4)-glucanohydrolase, 1,3-1 ,4-α-D-glucan 4-glucanohydrolase,1,4-α-D-glucan glucanehydrolase, 1,4-α-D-glucan glucohydrolase,1,4-(1,3:1,4)-β-D-glucan 4-glucanohydrolase, 1,4-β-D-glucanglucohydrolase, 1,4-β-D-xylan xylanohydrolase, 1,4-β-D-mannanmannanohydrolase, 1,5 -α-L-arabinanohydrolase, 1,4-α-D-glucanmaltohydrolase, 1,6-α-D-glucan 6-glucanohydrolase, 2,6-β-fructanfructanohydrolase, α-dextrin 6-glucanohydrolase, α-D-galactosidegalactohydrolase, α-D-glucoside glucohydrolase, α-D-mannosidemannohydrolase, acylneuraminyl hydrolase,Aerobacter-capsular-polysaccharide galactohydrolase,β-D-fructofuranoside fructohydrolase, β-D-fucoside fucohydrolase,α-D-fructan fructohydrolase, β-D-galactoside galactohydrolase,β-D-glucoside glucohydrolase, β-D-glucuronoside, glucuronosohydrolase,β-D-mannoside mannohydrolase, β-N-acetyl-D-hexosaminideN-acetylhexosamino hydrolase, cellulose-sulfate sulfohydrolase,collagenase, dextrin 6-α-D-glucanohydrolase,glycoprotein-phosphatidylinositol phosphatidohydrolase, hyaluronate4-glycanohydrolase, hyaluronoglucuronidase, pectin pectylhydrolase,peptidoglycan N-acetylmuramoylhydrolase, phosphatidylcholine2-acylhydrolase, phosphatidylcholine 1-acylhydrolase,poly(1,4-α-D-galacturonide),poly(1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolase, proteases,sucrose α-glucosidase, triacylglycerol acylhydrolase, triacylglycerolprotein-acylhydrolase.

The methods further can comprise administering at least one of anacid-stable subtilisin and an acid-stable DNAse I in an amount and for atime sufficient to cause significant biofilm degradation within of themammal. The methods can also comprise: identifying the presence of atleast one of Clostridium ssp, Klebsiella ssp, Pseudomonas ssp,Bacteroides ssp, Enterococcus ssp, Campylobacter ssp, Bacillus ssp,Yersinia ssp, Brucella ssp, Salmonella ssp, Shigella ssp, Fusobacteriumssp, Spirochaetes ssp, Entamoeba ssp, Candida ssp, Escherichia coli,Vibrio cholerae, Staphylococcus ssp, Streptococcus ssp, Hemophilus ssp,Aspergillus ssp and Gardnerella ssp in the mammal, and, administering tothe mammal a therapeutically effective amount of the anti-biofilmSerratia peptidase agent for a time sufficient to treat the identifiedmicroorganism.

The methods can comprise administering, or not administering, anantibiotic in conjunction with the Serratia peptidase, and otherpossible elements of the composition as discussed herein. The methodsfurther can also comprise administering one or more of an quercetin,seaprose or Fusarium protease in an amount and for a time sufficient tocause significant biofilm degradation within of the mammal.

In a further aspect, the methods comprise inhibiting at least one ofbacterial vaginosis, bacterial vaginitis or fungal vaginitis in amammal, the methods comprising: identifying the presence of the at leastone of bacterial vaginosis, bacterial vaginitis or fungal vaginitis,administering to the mammal a therapeutically effective amount of acomposition comprising at least one pharmaceutically acceptable carrierand Serratia peptidase, in an amount capable of significant reduction ofthe bacterial vaginosis, bacterial vaginitis or fungal vaginitis in themammal, in an amount and for a time sufficient to cause significantbacterial vaginosis, bacterial vaginitis or fungal vaginitis reductionwithin the mammal. In another aspect, the methods comprise inhibitingotitis media in a mammal, the methods comprising: identifying thepresence of the otitis media, administering to the mammal atherapeutically effective amount of a composition comprising at leastone pharmaceutically acceptable carrier and Serratia peptidase, in anamount capable of significant reduction of the otitis media in themammal, in an amount and for a time sufficient to cause significantotitis media reduction within the mammal. In yet another aspect, themethods comprise inhibiting osteomyelitis in a mammal, the methodscomprising: identifying the presence of the osteomyelitis, administeringto the mammal a therapeutically effective amount of a compositioncomprising at least one pharmaceutically acceptable carrier and Serratiapeptidase, in an amount capable of significant osteomyelitis reductionin the mammal, in an amount and for a time sufficient to causesignificant osteomyelitis reduction within the mammal. And, in a furtheraspect, the methods comprise inhibiting a biofilm on a mucosal surfacein a mammal, the method comprising: identifying the presence of thebiofilm on a mucosal surface, administering to the mammal atherapeutically effective amount of a composition comprising at leastone pharmaceutically acceptable carrier and Serratia peptidase, in anamount capable of significant biofilm reduction on the mucosal surfacein the mammal, in an amount and for a time sufficient to causesignificant biofilm reduction on the mucosal surface within the mammal.Such methods can further comprise one or more of the other featuresdiscussed herein.

These and other aspects, features, and embodiments are set forth withinthis application, including the following Detailed Description. Unlessexpressly stated otherwise, all embodiments, aspects, features, etc.,can be mixed and matched, combined, and permuted in any desired manner.

DETAILED DESCRIPTION

Biofilms in mammals have been implicated in a variety of possiblediseases, either as causing such diseases or making them worse. Thepresent compositions, systems, methods, etc., are directed to thereduction of biofilm(s) in certain sites and/or associated with certaindiseases or disorders in animals, including bacterial vaginosis,bacterial vaginitis or fungal vaginitis; osteomyelitis; otitis media;chronic sinusitis; biofilm on a mucosal surface; and, biofilm infectionsof medical implants and medical devices. The methods include inhibiting,treating, or reducing biofilms in such locations.

Exemplary Enzymes That Treat, Inhibit, Etc., Biofilms

Enzymes that disrupt the biofilm matrices of these organisms within thegastrointestinal tract or other target area are the subject of themethods, etc., herein.

Serratia peptidase

Serratia peptidase, also known as serrapeptase, is a known enzyme thatis an extracellular metalloprotease produced by Serratia sp.E15.Serratia peptidase is known to be absorbed in the GI tract. Serratiapeptidase can be enterically coated for example as tablets, andformulations including monovalent alginate. Tablets of Serratiapeptidase (5 mg/tablet) are marketed as Danzen or Dasen™ (TakedaChemical Industries, Ltd.), Aniflazym® (Takeda Pharma GmbH) and Serodase(Hayat Pharmaceutical Industries, Ltd.) Serratia peptidase may have beensold for use as an anti-inflammatory, analgesic and mucolytic.

Bromelain & Papain

Bromelain and papain are known enzymes and are known to be active withinthe gastrointestinal tract. Oral bromelain may have been reported toinhibit enterotoxigenic E. Coli attachment to the small intestine inpiglets, possibly by modifying receptor attachment sites. Bromelain isabsorbed through the intestine and it has anti-edema, anti-inflammatoryand anti-coagulation effects. Oral bromelain has been reported toattenuate inflammation in a murine model of asthma and to inhibit lungmetastases.

Phlogenzym® (Mucos Pharma GmbH & Co.) includes bromelain (90 mg),trypsin (48 mg) and the antioxidant flavonoid, rutin (100 mg) and hasbeen reported to reduce inflammation and experimental allergicencephalomyelitis. Wobenzym® N is reportedly a combination of bromelain,papain, trypsin, chymotrypsin, and rutin. Wobe-Mugos®, reportedlycontains trypsin, chymotrypsin and papain, has been reported to be aseffective as acyclovir in the treatment of herpes zoster.

Lactoferrin

Lactoferrin is also known as lactotransferrin. It is anaturally-occurring molecule, and is an extracellular iron-bindingglycoprotein which can be found in mucosal secretions, including thosefound in the respiratory tract, gastrointestinal tract, and urogenitaltract. It is also released by neutrophils at sites of infection. Duringinfection, the binding of iron by lactoferrin is proposed to reduce theamount of free extracellular iron. This process, known as thehypoferremia of infection, is thought to further limit the free ironavailable to invading microorganisms. Lactoferrin can be absorbedthrough the intestines. Enteric-formulated lactoferrin is moreefficiently absorbed from the intestine than is non-enteric-formulatedlactoferrin.

To applicant's knowledge, lactoferrin has not been combined withSerratia peptidase, bromelain or papain.

Otitis media

Otitis media has been described as an inflammation of the middle earthat is common in children.

Vaginosis

Bacterial vaginosis (BV) is a common lower genital tract syndromeaffecting women of reproductive age. BV is associated with adverseoutcomes among nonpregnant and pregnant women. BV has been found to beassociated with preterm labor, preterm delivery, low birth weight,postcesarean endometritis, and postabortion pelvic inflammatory disease.BV occurs when there are changes of the normal flora of the vagina,causing an increased prevalence of Gardnerella vaginalis, Mycoplasmahominis, and anaerobic organisms and a decreased prevalence of thenormally predominant Lactobacillus species. Previous studies have shownthat the alteration of the normal flora may increase the risk ofacquiring BV, HIV type 1, or other sexually transmitted diseases.

To applicant's knowledge, treatments recommended by the Centers forDisease Control (CDC) for BV include metronidazole or clindamycinadministered orally or intravaginally. Metronidazole is a nitroimidazolewith activity against anaerobic organisms, while clindamycin, amacrolide, has a broad spectrum of activity against a variety ofmicrobes including aerobic and anaerobic organisms. The CDC recommendsoral metronidazole for 7 days or vaginal metronidazole gel for 5 days,as they are equally effective. Metronidazole offers average cure ratesof 80% to 90%. Also, metronidazole is thought to be most effective fortreating infection that has spread into the upper reproductive tract.The CDC also recommends clindamycin cream 2% for 7 days, while notingthat it might not be as effective as metronidazole.

Despite treatment with either metronidazole or clindamycin, similarpercentages of women (approximately 10 to 15%) fail therapy after 1month. The proportion of women who relapse also increases over time. Therecurrence rate of BV is approximately 30% at 3 months and approximately50 to 80% at 1 year following therapy with either drug. Clindamycin'srelapse rate is higher: 4 weeks after clindamycin treatment, 56% ofwomen have recurring bacterial vaginosis. Current therapy for managingrecurrent BV is repeated treatment with antibiotics. An obvious problemand important health issue associated with repeated exposure to the sameantibiotic is resistance of those microbes targeted by the drug, whichcan result in an alteration of flora and possible persistence ofBV-associated pathogens.

Recent studies have shown an emergence of clindamycin-resistant genitalorganisms among clinically relevant bacteria, including group Bstreptococci. Resistance of BV to oral metronidazole has been postulatedto result from the adherent G. vaginalis biofilm that persists afterstandard therapy. In vitro models for G. vaginalis biofilm have beendeveloped.

Orthopedic Implants and Medical Devices

Implant-related infections are difficult to treat with antimicrobialagents alone. Several groups have reported on the properties ofimplant-associated biofilms and on the need for ancillary or adjuncttherapies.

The compositions and methods herein may not only treat but may inhibitor prevent implant-related infections via chronic administration of safeand effective oral compositions without antibiotics.

Other Enzymes and Molecules

In one aspect the suitable, physiologically acceptable anti-biofilmcompositions, etc., herein further comprise an amount of quercetin,seaprose (also known as seaprose-S) and/or Fusarium protease inconjunction with the Serratia peptidase, bromelain, papain and afibrinolytic enzyme, in an amount and for a time sufficient to causesignificant biofilm degradation within of the mammal.

Quercetin is an anti-inflammatory bioflavonoid. The composition isclaimed for the treatment of non-bacterial cystitis. It has beenreported that quercetin alone does not have a high bioavailability dueto the fact that transmural intestinal absorption is relatively low.

Seaprose, also known as Protease S or Seaprose S, is a semi-alkalineserine-proteinase produced by the fungus Aspergillus melleus. Seaprose-Sreportedly demonstrates an ability to reduce painful inflammation andbreak up mucus.

In one aspect the suitable, physiologically acceptable anti-biofilmcompositions, etc., herein further comprise an amount of anti-polymericβ-1,6-N-acetyl-D-glucosamine (poly-β-1,6-GlcNAc) agents to substantiallydisperse poly-β-1,6-GlcNAc and thus capable of significant biofilmdegradation. E.g., see Itoh Y, Wang X, Hinnebusch B J, Preston J F,Romeo T. Depolymerization of β-1,6-N-acetyl-D-glucosamine disrupts theintegrity of diverse bacterial biofilms. J Bacteriol 2005;187;382-7) Insome embodiments, for this and other agents, either alone or incombination, such significant reduction means, if measured in vitro, alog reduction of 1, typically 1.5, or 3.0-3.8 or better. In vivo, suchsignificant reduction can be substantial reduction of one or moresymptoms associated with a biofilm infection, or even substantialelimination of one or more symptoms associated with a biofilm infection.Exemplary anti-GlcNAc-agents include a previously identifiedβ-hexosaminidase and biofilm-dispersing enzyme of A.actinomycetemcomitans, DspB or dispersin B, which specificallyhydrolyzes the glycosidic linkages of poly-β-1,6-GlcNAc and disruptsbacterial biofilm (Kaplan J B, Ragunath C, Ramasubbu N, Fine D H. 2003.Detachment of Actinobacillus actinomycetemcomitans biofilm cells by anendogenous β-hexosaminidase activity. J Bacteriol 2003;185:4693-8).Dispersin B cleaves β(1,6)-linked N-acetylglucosamine polymer using acatalytic machinery similar to other family 20 hexosaminidases whichcleave β(1,4)-linked N-acetylglucosamine residues. Dispersin B andsimilar hexosaminidases with activity in biofilms are suitable for usein the methods, physiologically acceptable anti-biofilm compositions,etc., discussed herein. The anti-poly-β-1,6-GlcNAc agents can be usedwith, or instead of, cellulase, discussed further below, althoughtypically they are used together.

In one aspect the suitable, physiologically acceptable anti-biofilmcompositions comprise a cellulase in an amount capable of significantbiofilm degradation. Such cellulases can have activity against, forexample, cellulose in a salmonella biofilm or others. Cellulase refersto a class of enzymes produced chiefly by fungi, bacteria, andprotozoans that catalyze the hydrolysis of cellulose. However, there arealso cellulases produced by other types of organisms such as plants andanimals. Cellulases that have been used as digestive enzymes are knownto be acid-stable. These include but are not limited to cellulases fromAspergillus species. Several different kinds of cellulases are known,which differ structurally and mechanistically. The EC number for thisgroup of enzymes is EC 3.2.1.4. The reaction catalyzed is theendohydrolysis of 1,4-β-D-glycosidic linkages in cellulose. Other namesfor cellulase are: Endoglucanase, endo-1,4-β-glucanase, carboxymethylcellulose, endo-1,4-β-D-glucanase, β-1,4-glucanase, β-1,4-endoglucanhydrolase, celludextrinase, avicelase. Cellulases have been used invitro in the disruption of biofilms on medical implants under acidic pHconditions (Loiselle M, Anderson K W, The use of cellulase in inhibitingbiofilm formation from organisms commonly found on medical implants.Biofouling 2003;19:77-85.) In typical embodiments, the cellulase(s)herein are resistant to denaturation/inactivation at a pH range of 1.0to 5.0 and 10 to 14, possesses hydrolytic activity across a pH span of 1to 14, has effective hydrolytic activity within the gastric environmentat a fasting pH of 1.0 to 3.0 and in the presence of food and otheringested material, and/or possesses effective hydrolytic activity at apH of 4.5 to 7.5 encompassing physiologic pH in the small intestines andcolon.

Commercial sources of cellulases, hemicellulases and other enzymes thatmay be used include the following: Deerland Enzymes, Kennesaw, Ga.(www.deerlandenzymes.com); National Enzyme Company(www.nationalenzyme.com), Specialty Enzymes (www.specialtyenzymes.com);and others. The enzymes may be derived from any suitable source such asplant, bacterial, fungal or animal sources.

In one embodiment, the anti-biofilm compositions herein further comprisephysiologically acceptable cellulase, hemicellulase/pectinase complex,β-gluconase, acid protease, and alkaline protease, with at least onepharmaceutically acceptable carrier, diluents, excipients, buffers, oradjuvants. Pharmaceutically acceptable carriers or diluents, excipients,buffers, adjuvants, and the like are nontoxic to recipients at thedosages and concentrations employed.

In one embodiment, the amount of cellulase per oral dose is about100-300 CU, and typically about 200 CU; the amount ofhemicellulase/pectinase complex is about 60-100 HSU, and typically about80 HSU; the amount of β-gluconase is about 6-10 BGU, and typically about8 BGU; the amount of acid protease is about 15-25 SAP, and typicallyabout 20 SAP; and, the amount of alkaline protease is about 15-25 HUT,and typically about 20 HUT.

In still further embodiments, the amount of cellulase per oral doseranges from 1 to 10,000 CU, the amount of hemicellulase/pectinasecomplex ranges from 1 to 8,000 HSU, the amount of β-gluconase rangesfrom 1 to 1000 BGU, the amount of acid protease ranges from 1 to 10,000SAP, and the amount of alkaline protease ranges from 1 to 40,000 HUT.

In a further embodiment, the physiologically acceptable anti-biofilmcomposition comprises cellulase, hemicellulase/pectinase complex,β-gluconase, acid protease, alkaline protease, and any one or more ofthe following in an amount capable an amount capable of significantbiofilm degradation: disaccharides, amylase, -amylase, β-amylase,glucoamylase, endoglucanase, xylanase, lipase, lysozyme, any enzyme suchas a protease, peptidase or protease/peptidase complex with dipeptidylpeptidase IV (DPP-IV) activity, chitosanase, bromelain, papain, ficin,kiwi protease, any plant-derived protease or proteinase, or phytase.

In a further embodiment, the physiologically acceptable anti-biofilmcomposition is composed of cellulase, hemicellulase/pectinase complex,β-gluconase, acid protease, alkaline protease, and any one or more ofthe following specific enzymes in an amount capable of biofilmdegradation: 1,2-1,3-

-D-mannan mannohydrolase, 1,3-β-D-xylanxylanohydrolase, 1,3-β-D-glucanglucanohydrolase, 1,3(1,3;1,4)-

-D-glucan 3-glucanohydrolase, 1,3(1,3;1,4)-β-D-glucan3(4)-glucanohydrolase, 1,3-1,4-

-D-glucan 4-glucanohydrolase, 1,4-

-D-glucan glucanehydrolase, 1,4-

-D-glucan glucohydrolase, 1,4-(1,3:1,4)-β-D-glucan 4-glucanohydrolase,1,4-β-D-glucan glucohydrolase, 1,4-β-D-xylan xylanohydrolase,1,4-β-D-mannan mannanohydrolase, 1,5-

-L-arabinanohydrolase, 1,4-

-D-glucan maltohydrolase, 1,6-

-D-glucan 6-glucanohydrolase, 2,6-β-fructan fructanohydrolase,

-dextrin 6-glucanohydrolase,

-D-galactoside galactohydrolase,

-D-glucoside glucohydrolase,

-D-mannoside mannohydrolase, acylneuraminyl hydrolase,Aerobacter-capsular-polysaccharide galactohydrolase,β-D-fructofuranoside fructohydrolase, β-D-fucoside fucohydrolase,

-D-fructan fructohydrolase, β-D-galactoside galactohydrolase,β-D-glucoside glucohydrolase, β-D-glucuronoside, glucuronosohydrolase,β-D-mannoside mannohydrolase, β-N-acetyl-D-hexosaminideN-acetylhexosamino hydrolase, cellulose-sulfate sulfohydrolase,collagenase, dextrin 6-

-D-glucanohydrolase, glycoprotein-phosphatidylinositolphosphatidohydrolase, hyaluronate 4-glycanohydrolase,hyaluronoglucuronidase, pectin pectylhydrolase, peptidoglycanN-acetylmuramoylhydrolase, phosphatidylcholine 2-acylhydrolase,phosphatidylcholine 1-acylhydrolase, poly(1,4-

-D-galacturonide),poly(1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolase, proteases,sucrose

-glucosidase, triacylglycerol acylhydrolase, triacylglycerolprotein-acylhydrolase.

Another group of enzymes that may be employed in the methods, etc.herein is a sub-group of serine proteases commonly designated assubtilisins. A subtilisin is a serine protease produced by Gram-positivebacteria or fungi. The amino acid sequences of a number of subtilisinshave been determined, including at least six subtilisins from Bacillusstrains, namely, subtilisin 168, subtilisin BPN, subtilisin Carlsberg,subtilisin DY, subtilisin amylosacchariticus, and mesentericopeptidase,one subtilisin from an actinomycetales, thermitase fromThermoactinomyces vulgaris, and one fungal subtilisin, proteinase K fromTritirachium album.

An exemplary lipase as discussed above can be a microbial lipase. Assuch, the lipase may be selected from yeast lipases, e.g., Candida, andbacterial lipases, e.g., Pseudomonas or Bacillus, lipases; or fungal,e.g., Humicola or Rhizomucor.

Examples of amylases useful in the methods, etc., herein includeBacillus amylases, e.g., Bacillus stearothermophilus amylase, Bacillusamyloliquefaciens amylase, Bacillus subtilis amylase or Bacilluslicheniformis amylase or Aspergillus amylases, e.g., Aspergillus nigeror Aspergillus oryzae amylase.

Another group of enzymes useful in the methods, etc., herein includepectinases belonging to the enzyme classes polygalacturonases(EC3.2.1.15), pectinesterases (EC3.2.1.11), pectin lyases (EC4.2.2.10)and hemicellulases such as endo-1,3-β-xylosidase (EC 3.2.1.32), xylan1,4-β-xylosidase (EC 3.2.1.37) and -L-arabinofuranosidase (EC 3.2.1.55).A suitable source organism for pectinases may be Aspergillus niger orAspergillus aculeatus.

Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase,is a 14.4 kilodalton enzyme (EC 3.2.1.17) that damages bacterial cellwalls by catalyzing hydrolysis of 1,4-β-linkages between N-acetylmuramicacid and N-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is found insaliva, tears, and polymorphonucleocytes and has known antibacterialactivity. The enzyme functions by attacking peptidoglycans (found in thecells walls of bacteria, especially Gram-positive bacteria) andhydrolyzing the glycosidic bond that connects N-acetylmuramic acid withthe fourth carbon atom of N-acetylglucosamine. Lysozyme has been used inthe treatment of otitis media and sinusitis (U.S. Pat. No. 7,060,674).Oral lysozyme compositions have been used in the treatment of variousconditions in humans, including arthritis (U.S. Pat. No. 7,229,809).

Another enzyme that may be employed in the methods, etc. herein isdeoxyribonuclease I (DNase I), a phosphodiesterase capable ofhydrolyzing polydeoxyribonucleic acid. DNase I has been purified fromvarious species to various degrees. DNase I, when inhaled, affects thecapability of P. aeruginosa to form biofilms in the lungs in the initialdevelopment stages. DNase I hydrolyzes the DNA present in sputum/mucusof cystic fibrosis patients and reduces viscosity in the lungs,promoting improved clearance of secretions. Enzymes that are acid-stableare candidates for use in conjunction with the methods, physiologicallyacceptable anti-biofilm compositions, etc., discussed herein. DNase Iactivities are classifiable into three groups on the basis of theirdifferent tissue distributions of DNase I. DNase I of parotid type issecreted from the parotid gland and must pass through the very acidicconditions in the stomach.

The physiologically acceptable anti-biofilm compositions, methods, etc.,herein are to be taken by mouth, typically at least 1 hour before or 42hours after a meal or consumption of food. The physiologicallyacceptable anti-biofilm compositions, methods, etc., herein aretypically to be taken 2 to 4 times per day (other intervals may beappropriate in certain circumstances) and the regimen is typically to befollowed for an extended period, for example at least about 1 or 2months.

The enzyme preparations may be combined with a natural antimicrobialsuch as oil of oregano, berberine, or undecylenic acid or with aprescription antibiotic or antimicrobial.

The enzyme preparations may be combined with the oral intake of one ormore probiotic microorganisms or prebiotic compositions. For example,such preparations can be consumed in the same composition as a probioticmicroorganisms or prebiotic composition, simultaneously with suchprobiotic microorganisms or prebiotic compositions, or separately but inconjunction with such probiotic microorganisms or prebioticcompositions. The World Health Organization defines probiotic organismsas live microorganisms that when administered in adequate amounts confera health benefit on the host. The enzyme preparation may be combinedwith one or more prebiotics. A prebiotic is defined as “selectivelyfermented ingredients that allow specific changes, both in thecomposition and/or activity in the gastrointestinal microflora thatconfer benefits upon host well-being and health.” (Roberfroid M.Prebiotics: the concept revisited. J Nutr 2007;137(3 Suppl 2):830S-7S.)

Methods related to the compositions, etc., herein include methods ofscreening, making and using, including for the manufacture ofmedicaments.

In some aspects, the methods comprise inhibiting a biofilm infection ina mammal, for example gastrointestinal or at one of the other targetsites discussed herein, the method comprising: identifying the presenceof the biofilm infection, administering, orally or otherwise, to themammal a therapeutically effective amount of at least one anti-biofilmagent comprising at least one pharmaceutically acceptable carrier and atherapeutic amount of Serratia peptidase, in amounts capable ofsignificant biofilm degradation in the mammal upon administration to themammal. In further embodiments the methods comprise administering one ormore of bromelain, papain and a fibrinolytic enzyme with the Serratiapeptidase, and one or more of the other aspects or elements of thecompositions, etc., herein.

The compositions herein can be for use as an active therapeuticsubstance, for use in the manufacture of a medicament for inhibiting ortreating a gastrointestinal biofilm in a mammal, or for manufacturing amedicament able to reduce symptoms associated with a gastrointestinalbiofilm in a human patient, for example comprising combining apharmaceutically effective amount of and a therapeutic amount ofSerratia peptidase in an amount capable of significant biofilmdegradation with at least one of a pharmaceutically acceptable carrier,adjuvant, excipient, buffer and diluent. In further embodiments themethods further comprise administering one or more of bromelain, papainand a fibrinolytic enzyme with the Serratia peptidase,

Exemplary Biofilm Targets

Exemplary target biofilm organisms, including both indigenous andbiofilm infectious organisms are discussed below.

Enterococci

Enterococci, although part of the normal flora of the humangastrointestinal tract, have been recognized as an important cause ofnosocomial infection for over two decades and are commonly implicated inurinary tract infections, bacteremia, intra-abdominal and surgical woundinfections, catheter-related infections, and endocarditis.

Staphylococcus

Pathogenic staphylococci can form biofilms in which they show a higherresistance to antibiotics and the immune defense system than theirplanktonic counterparts. Staphylococcus aureus is a common pathogenassociated with nosocomial infections. It can persist in clinicalsettings and gain increased resistance to antimicrobial agents throughbiofilm formation. Staphylococcus aureus is among the leading pathogenscausing bloodstream infections able to form biofilms on host tissue andindwelling medical devices and to persist and cause disease. Infectionscaused by S. aureus are becoming more difficult to treat because ofincreasing resistance to antibiotics (e.g., vancomycin ormethicillin-resistant Staphylococcus aureus). In a biofilm environmentparticularly, microbes exhibit enhanced resistance to antimicrobialagents.

Pseudomonas

The human opportunistic pathogen, Pseudomonas aeruginosa, is a majorcause of infectious-related mortality among the critically ill patients,and carries one of the highest case fatality rates of all gram-negativeinfections. Although the lungs have been traditionally considered to bea major site of P. aeruginosa infection among critically ill patients, asignificant number of these infections arise as a result of directcontamination of the airways by the gastrointestinal flora or byhematogenous dissemination from the intestine to the lung parenchyma.Pseudomonas aeruginosa causes severe infections in immunologicallycompromised patients and is a major pathogen in cystic fibrosispatients. An important virulence mechanism is the formation of a mucoidbiofilm. Secreted alginate is a crucial constituent of the mucoidbiofilm matrix. However, alginate-negative mutants of P. aeruginosa arealso able to form nonmucoid biofilms, showing an architecture differentfrom that of biofilms formed by alginate-overproducing mucoid P.aeruginosa (Nivens D E, Ohman D E, Williams J, Franklin M J. Role ofalginate and its O acetylation in formation of Pseudomonas aeruginosamicrocolonies and biofilms. J Bacteriol 2001;183:1047-57; Wozniak D J,Wyckoff T J, Starkey M, Keyser R, Azadi P, O'Toole G A, Parsek M R.Alginate is not a significant component of the extracellularpolysaccharide matrix of PA14 and PAO1Pseudomonas aeruginosa biofilms.Proc Natl Acad Sci USA 2003;100:7907-12.)

Helicobacter pylori

H. pylori is one of the more common human pathogens infecting 50% of theworld's population. It is associated with duodenal ulcers, gastriculcers, gastritis, and gastric carcinoma. Treatment of H. pylori isdifficult involving multidrug regimens and lengthy treatment periods.There is a 10-20% relapse rate. Recent studies document the importanceof biofilms in the pathogenesis of H. pylori disease. (Coticchia J M etal. Presence and density of Helicobacter pylori biofilms in humangastric mucosa in patients with peptic ulcer disease. J GastrointestSurg. 2006;10:883-9) An oral multienzyme formulation holds great promiseto facilitate the elimination of H. pylori biofilm and the eradicationof H. pylori pathogens thereby reducing the risk of gastritis, pepticulcer disease, and gastric cancer.

Listeria

The foodborne pathogen Listeria is the causative agent of listeriosis, asevere disease where the overt form has a severe mortality greater than25%. Listeria monocytogenes can survive and grow over a wide range ofenvironmental conditions such as refrigeration temperatures, low pH andhigh salt concentration. This allows the pathogen to overcome foodpreservation and safety barriers, and pose a potential risk to humanhealth. Listeria monocytogenes may specifically be found in raw foods,such as unpasteurized fluid milk, raw vegetables, raw and cookedpoultry. It has the ability to grow at low temperatures; thus, allowingit to grow in refrigerated foods. Listeria monocytogenes was thought tobe exclusively associated as infections in animals, but recently, thispathogenic species has also been isolated, in its dormant form, in theintestinal tract of small percentage of the human population (RouquetteC, Berche P. The pathogenesis of infection by Listeria monocytogenes.Microbiologia 1996;12:245-58).

Campylobacter

Campylobacter jejuni is a species of curved, rod-shaped, Gram-negativemicroaerophilic, bacteria commonly found in animal feces. It is one ofthe most common causes of human gastroenteritis in the world. Foodpoisoning caused byCampylobacter species can be severely debilitatingbut is rarely life-threatening. It has been linked with subsequentdevelopment of Guillain-Barré syndrome (GBS), which usually develops twoto three weeks after the initial illness. Contaminated food is a majorsource of isolated infections, with incorrectly prepared meat andpoultry normally the source of the bacteria. Infection with C. jejuniusually results in enteritis, which is characterized by abdominal pain,diarrhea, fever, and malaise. The major gastrointestinal pathogenCampylobacter jejuni is shown to exist as three forms of monospeciesbiofilm in liquid culture. (Joshua G W, Guthrie-Irons C, Karlyshev A V,Wren B W. Biofilm formation in Campylobacter jejuni. Microbiology 2006;152(Pt 2):387-96.)

Bacillus anthracis

Bacillus anthracis is a Gram-positive, endospore-forming bacterium andis the aetiological agent of pulmonary, gastrointestinal and cutaneousanthrax. In endemic areas in which humans and livestock interact,chronic cases of cutaneous anthrax are commonly reported. Currently,there are few data known to the inventor that account for the importanceof the biofilm mode of life in B. anthracis, yet biofilms have beencharacterized in other pathogenic and non-pathogenic Bacillus species,including Bacillus cereus and Bacillus subtilis, respectively. B.anthracis readily forms biofilms which are inherently resistant tocommonly prescribed antibiotics. (Lee K, Costerton J W, Ravel J,Auerbach R K, Wagner D M, Keim P, Leid J G. Phenotypic and functionalcharacterization of Bacillus anthracis biofilms. Microbiology2007;153(Pt 6):1693-701.)

Yersinia

Yersiniosis is an infectious disease caused by a bacterium of the genusYersinia. In the United States, most human illness is caused by onespecies, Y. enterocolitica. Infection with Y. enterocolitica occurs mostoften in young children. Common symptoms in children are fever,abdominal pain, and diarrhea. Gastrointestinal symptoms are common inboth the acute and chronic states of yersiniosis. Infection is mostoften acquired by eating contaminated food, especially raw orundercooked pork products. Drinking contaminated unpasteurized milk oruntreated water can also transmit the infection.

Yersinia pestis, the causative agent of bubonic plague, is transmittedto rodents and humans by the bites of fleas whose proventriculi areblocked by a dense mass of the biofilm bacteria. (Tan L, Darby C. Amovable surface: formation of Yersinia sp. biofilms on motileCaenorhabditis elegans. J Bacteriol. 2004;186:5087-92.) The blockagestarves the flea and stimulates it to bite repeatedly in search of bloodmeals, thus spreading the bacteria to new hosts. Biofilm models usingCaenorhabditis elegans may be used to identify enzymes that killYersinia biofilms (Styer K L, Hopkins G W, Bartra S S, Plano G V,Frothingham R, Aballay A. Yersinia pestis kills Caenorhabditis elegansby a biofilm-independent process that involves novel virulence factors.EMBO reports 2005;10:992-7.)

Brucella species

Humans are generally infected in one of three ways: eating or drinkingsomething that is contaminated with Brucella, breathing in the organism(inhalation), or having the bacteria enter the body through skin wounds.The most common way to be infected is by eating or drinking contaminatedmilk products.

Salmonella

Salmonella enterica, a foodborne pathogen that causes salmonellosis, iscaused by the ingestion of bacteria that invade the intestinalepithelium and multiply there. Salmonella enterica is known to formbiofilms, and its attachment to, and growth on, eukaryotic cells isfacilitated by exopolysaccharides (Ledeboer & Jones, 2005). Most personsinfected with Salmonella develop diarrhea, fever, and abdominal cramps12 to 72 hours after infection. The illness usually lasts 4 to 7 days,and most persons recover without treatment. However, in some persons thediarrhea may be so severe that the patient needs to be hospitalized. Inthese patients, the Salmonella infection may spread from the intestinesto the blood stream, and then to other body sites and can cause deathunless the person is treated promptly.

Shigella

There are several different kinds of Shigella bacteria: Shigella sonnei,also known as “Group D” Shigella, accounts for over two-thirds of theshigellosis in the United States. Shigellosis is an infectious diseasecaused by a group of bacteria called Shigella. Most who are infectedwith Shigella develop diarrhea, fever, and stomach cramps starting a dayor two after they are exposed to the bacterium. Some Shigella bacteriahave become resistant to antibiotics. A second type, Shigella flexneri,or “group B” Shigella, accounts for almost all of the rest. Other typesof Shigella continue to be important causes of disease in the developingworld. One type found in the developing world, Shigella dysenteriae type1,causes deadly epidemics there.

Typhi (typhoid fever)

Salmonella enterica serovar Typhi causes typhoid fever, an enteric feverthat is potentially fatal. Asymptomatic carriers may carry bacteria inthe gallbladder. Salmonella typhi lives only in humans. Persons withtyphoid fever carry the bacteria in their bloodstream and intestinaltract. In addition, a small number of persons, called carriers, recoverfrom typhoid fever but continue to carry the bacteria. Both ill personsand carriers shed S. typhi in their feces (stool). Salmonella typhi istransmitted in contaminated food, water and beverages. A system wasrecently developed to analyze salmonella biofilm formation on glasscoverslips (Prouty A M, Schwesinger W H, Gunn J S. Biofilm formation andinteraction with the surfaces of gallstones by Salmonella spp. InfectImmun 2002;70:2640-9.)

Escherichia coli

Enterotoxigenic Escherichia coli targets the small intestine where thebarrier effect of the autochthonous microflora is low due to higheracidity and peristaltic movements in this region. This organism adheresto and colonizes the mucus in order to elicit a pathogenic effect(Knutton S, Lloyd D R, Candy D C, McNeish A S. In vitro adhesion ofenterotoxigenic Escherichia coli to human intestinal epithelial cellsfrom mucosal biopsies. Infect Immun 1984;44:514-8.) This means that thepathogen and/or its toxins can readily adhere to exposed eneterocytesand invade the host.

Vibrio cholerae (cholera)

Vibrio cholerae is a Gram-negative, facultative pathogen that is thecausative agent of cholera, a devastating diarrheal disease that affectsmillions of people in the developing world each year; it survives inaqueous reservoirs, probably in the form of biofilms.

Entamoeba histolytica

Invasive intestinal amebiasis, caused by Entamoeba histolytica, isinitiated with attachment of trophozoites to the colonic mucous layer,mucous disruption and/or depletion, and adherence to and cytolysis ofhost epithelial and inflammatory cells. A current working model ofintestinal amebiasis suggests that the microenvironment of the hostintestine, particularly intestinal mucins and the bacterial biofilm, mayinfluence the behavior of pathogenic amebae. Enzymes that disruptbacterial biofilm will be useful in the inhibition and treatment ofamebiasis.

All terms used herein, are used in accordance with their ordinarymeanings unless the context or definition clearly indicates otherwise.Also unless expressly indicated otherwise, the use of “or” includes“and” and vice-versa. Non-limiting terms are not to be construed aslimiting unless expressly stated, or the context clearly indicates,otherwise (for example, “including,” “having,” and “comprising”typically indicate “including without limitation”). Singular forms,including in the claims, such as “a,” “an,” and “the” include the pluralreference unless expressly stated, or the context clearly indicates,otherwise.

The scope of the present physiologically acceptable anti-biofilmcompositions, systems and methods, etc., includes both means plusfunction and step plus function concepts. However, claims are not to beinterpreted as indicating a “means plus function” relationship unlessthe word “means” is specifically recited in a claim, and are to beinterpreted as indicating a “means plus function” relationship where theword “means” is specifically recited in a claim. Similarly, claims arenot to be interpreted as indicating a “step plus function” relationshipunless the word “step” is specifically recited in a claim, and are to beinterpreted as indicating a “step plus function” relationship where theword “step” is specifically recited in a claim.

From the foregoing, it will be appreciated that, although specificembodiments have been discussed herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of the discussion herein. Accordingly, the systems and methods,etc., include such modifications as well as all permutations andcombinations of the subject matter set forth herein and are not limitedexcept as by the appended claims or other claim having adequate supportin the discussion herein.

1-92. (canceled)
 93. A method of inhibiting sinusitis in a mammal, the method comprising: identifying the presence of the sinusitis, and administering to the mammal a therapeutically effective amount of a composition comprising at least one pharmaceutically acceptable carrier and Serratia peptidase, in an amount capable of significant reduction of the sinusitis in the mammal, in an amount and for a time sufficient to cause significant sinusitis reduction within the mammal.
 94. The method of claim 93 wherein the method further comprises administering at least one of bromelain, papain and a fibrinolytic enzyme for a time sufficient to cause significant reduction of the sinusitis in the mammal.
 95. The method of claim 94 wherein the fibrinolytic enzyme comprises at least one of nattokinase or lumbrokinase.
 96. The method of claim 93 wherein the method further comprises administering all of bromelain, papain and a fibrinolytic enzyme for a time sufficient to cause significant reduction of the sinusitis in the mammal.
 97. The method of claim 93 or 94 wherein the method further comprises administering at least one of lactoferrin and a chelating agent in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 98. The method of claim 93 or 94 wherein the method further comprises administering at least one of an anti-sinusitis acid-stable cellulase or an anti-sinusitis anti-polymeric β-1,6-N-acetyl-D-glucosamine (poly-β-1,6-GlcNAc) agent in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 99. The method of claim 93 or 94 wherein the method further comprises administering at least one of an acid-stable hemicellulase/pectinase complex, β-gluconase, acid protease, or alkaline protease in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 100. The method of claim 93 or 94 wherein the method further comprises administering at least one an acid-stable agent in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal, the at least one agent selected from the following: a disaccharidase; amylase; α-amylase; β-amylase; glucoamylase; endoglucanase; xylanase; lipase; lysozyme; an enzyme with dipeptidyl peptidase IV (DPP-IV) activity; chitosanase; ficin; kiwi protease; any plant-derived protease or proteinase, or phytase.
 101. The method of claim 93 or 94 wherein the method further comprises administering at least one an acid-stable enzyme in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal, the at least one enzyme selected from the following: 1,2-1,3-α-D-mannan mannohydrolase, 1,3-β-D-xylanxylanohydrolase, 1,3-β-D-glucan glucanohydrolase, 1,3(1,3;1,4)-α-D-glucan 3-glucanohydrolase, 1,3(1,3;1,4)-β-D-glucan 3(4)-glucanohydrolase, 1,3-1,4-α-D-glucan 4-glucanohydrolase, 1,4-α-D-glucan glucanehydrolase, 1,4-α-D-glucan glucohydrolase, 1,4-(1,3:1,4)-β-D-glucan 4-glucanohydrolase, 1,4-β-D-glucan glucohydrolase, 1,4-β-D-xylan xylanohydrolase, 1,4-β-D-mannan mannanohydrolase, 1,5-α-L-arabinanohydrolase, 1,4-α-D-glucan maltohydrolase, 1,6-α-D-glucan 6-glucanohydrolase, 2,6-β-fructan fructanohydrolase, α-dextrin 6-glucanohydrolase, α-D-galactoside galactohydrolase, α-D-glucoside glucohydrolase, α-D-mannoside mannohydrolase, acylneuraminyl hydrolase, Aerobacter-capsular-polysaccharide galactohydrolase, β-D-fructofuranoside fructohydrolase, β-D-fucoside fucohydrolase, α-D-fructan fructohydrolase, β-D-galactoside galactohydrolase, β-D-glucoside glucohydrolase, β-D-glucuronoside, glucuronosohydrolase, β-D-mannoside mannohydrolase, β-N-acetyl-D-hexosaminide N-acetylhexosamino hydrolase, cellulose-sulfate sulfohydrolase, collagenase, dextrin 6-α-D-glucanohydrolase, glycoprotein-phosphatidylinositol phosphatidohydrolase, hyaluronate 4-glycanohydrolase, hyaluronoglucuronidase, pectin pectylhydrolase, peptidoglycan N-acetylmuramoylhydrolase, phosphatidylcholine 2-acylhydrolase, phosphatidylcholine 1-acylhydrolase, poly(1,4-α-D-galacturonide), poly(1,4-(N-acetyl-β-D-glucosaminide))-glycanohydrolase, proteases, sucrose α-glucosidase, triacylglycerol acylhydrolase, triacylglycerol protein-acylhydrolase.
 102. The method of claim 93 or 94 wherein the method further comprises administering at least one of an acid-stable subtilisin and an acid-stable DNAse I in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 103. The method of claim 93 or 94 wherein the method further comprises: further identifying the presence of at least one of Clostridium ssp, Klebsiella ssp, Pseudomonas ssp, Bacteroides ssp, Enterococcus ssp, Campylobacter ssp, Bacillus ssp, Yersinia ssp, Brucella ssp, Salmonella ssp, Shigella ssp, Fusobacterium ssp, Spirochaetes ssp, Entamoeba ssp, Candida ssp, Escherichia coli, Vibrio cholerae, Staphylococcus ssp, Streptococcus ssp, Hemophilus ssp, Aspergillus ssp and Gardnerella ssp in the mammal, and administering to the mammal a therapeutically effective amount of the anti-biofilm Serratia peptidase agent for a time sufficient to treat the identified Clostridium ssp, Klebsiella ssp, Pseudomonas ssp, Bacteroides ssp, Enterococcus ssp, Campylobacter ssp, Bacillus ssp, Yersinia ssp, Brucella ssp, Salmonella ssp, Shigella ssp, Fusobacterium ssp, Spirochaetes ssp, Entamoeba ssp, Candida ssp, Escherichia coli, Vibrio cholerae, Staphylococcus ssp, Streptococcus ssp, Hemophilus ssp, Aspergillus ssp and Gardnerella ssp.
 104. The method of claim 93 or 94 wherein the method further comprises administering a lactoferrin peptide in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 105. The method of claim 93 or 94 wherein the method further comprises administering a green tea extract in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal.
 106. The method of claim 93 or 94 wherein the method further comprises administering in an amount and for a time sufficient to cause significant reduction of the sinusitis within of the mammal, a chelating agent selected from the group comprising ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA); the disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium and diammonium salts of EDTA; the barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, and zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraaceticacid monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3 -diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid; 1,3 -diaminopropane-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionic acid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid)hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid; ethylenediamine-N,N,N′,N′-tetrakis(methylenephosponic acid); O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid; N,N-bis(2-hydroxybenzyl)ethylene diamine-N,N-diacetic acid; 1,6-hexamethylenediamine-N,N,N′,N′-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2-diaminopropane-N,N,N′,N′-tetraacetic acid; nitrilotriacetic acid; nitrilotripropionic acid; the trisodium salt of nitrilotris(methylenephosphoric acid); 7,19,30-trioxa -1,4,10,13,16,22,27,33 -octaazabicyclo [11,11,11]pentatriacontane hexahydrobromide; triethylenetetraminie-N,N,N′,N″,N″′,N″′-hexaacetic acid; deferoxamine; deferiprone; and deferasirox.
 107. The method of claim 93 or 94 wherein the method further comprises administering an antibiotic in conjunction with the Serratia peptidase, bromelain, papain and a fibrinolytic enzyme, in an amount and for a time sufficient to cause significant biofilm degradation within of the mammal.
 108. The method of claim 93 or 94 wherein the method further comprises not administering an antibiotic in conjunction with the Serratia peptidase, bromelain, papain and a fibrinolytic enzyme, in an amount and for a time sufficient to cause significant biofilm degradation within of the mammal.
 109. The method of claim 93 or 94 wherein the method further comprises administering an quercetin in conjunction with the Serratia peptidase, bromelain, papain and a fibrinolytic enzyme, in an amount and for a time sufficient to cause significant biofilm degradation within of the mammal.
 110. The method of claim 93 or 94 wherein the method further comprises administering seaprose in conjunction with the Serratia peptidase, bromelain, papain and a fibrinolytic enzyme, in an amount and for a time sufficient to cause significant biofilm degradation within of the mammal.
 111. The method of claim 93 or 94 wherein the method further comprises administering Fusarium protease in conjunction with the Serratia peptidase, bromelain, papain and a fibrinolytic enzyme, in an amount and for a time sufficient to cause significant biofilm degradation within of the mammal. 112-130. (canceled) 